4&#39;-Deschlororebeccamycin pharmaceutical composition and method of use

ABSTRACT

A new antitumor antibiotic designated herein as 4&#39;-deschlororebeccamycin is produced by fermentation of Nocardia aerocolonigenes ATCC 39243. The new compound possesses antibacterial activity and inhibits the growth of tumors in experimental animals.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel antitumor antibiotic and to itsproduction and recovery.

2. Description of the Prior Art

The novel compound of the present invention is related in structure tothe antitumor agent, rebeccamycin, disclosed and claimed in co-pendingapplication Ser. No. 461,817 filed Jan. 28, 1983, the entire disclosureof which is hereby incorporated by reference. Rebeccamycin has theformula ##STR1## and is obtained by cultivating Nocardiaaerocolonigenes.

Somewhat related in structure to the compound of the present inventionis the antitumor agent, staurosporine (also called AM-2282), obtainedfrom fermentation of Streptomyces staurosporeus. Staurosporine isdescribed in J.C.S. Chem. Comm., 1978, Pg. 800-801 and in J. Antibiotics30(4): 275-282 (1977).

Agnew. Chem. Int. Ed. Engl. 19(6): 459-460 (1980) discloses severalindole pigments obtained from the fruiting bodies of the slime moldArcyria denudata which are structurally related to staurosporine.Certain of the pigments exhibit activity against Bacillus brevis and B.subtilis.

SUMMARY OF THE INVENTION

This invention relates to a new antitumor antibiotic designated hereinas 4'-deschlororebeccamycin having the structural formula ##STR2## andto the process for the preparation, isolation and purification of4'-deschlororebeccamycin in substantially pure form.

The antibiotic of the present invention is obtained by fermentation of a4'-deschlororebeccamycin-producing strain of Nocardia aerocolonigenes,preferably Nocardia aerocolonigenes strain C38,383-RK2 (ATCC 39243) or amutant thereof, in an aqueous nutrient medium under submerged aerobicconditions until a substantial amount of 4'-deschlororebeccamycin isproduced by said microorganism in said culture medium and, optionally,recovering the 4'-deschlororebeccamycin from the culture mediumsubstantially free of co-produced substances.

The compound 4'-deschlororebeccamycin exhibits antimicrobial activityand also activity against experimental animal tumor systems, e.g. P-388leukemia in mice.

DETAILED DESCRIPTION

The 4'-deschlororebeccamycin of the present invention is produced byfermentation of a 4'-deschlororebeccamycin-producing strain of Nocardiaaerocolonigenes.

An especially preferred 4'-deschlororebeccamycin-producing strain isthat disclosed in U.S. application Ser. No. 461,817 filed Jan. 28, 1983as being the producing organism for rebeccamycin. The present applicanthas discovered that during cultivation of this microorganism there isco-produced along with rebeccamycin the 4'-deschlororebeccamycin productof the present invention. This preferred producing microorganism,designated strain C38,383-RK2, was isolated from a soil sample collectedin Panama. Cultures of this strain have been deposited in the AmericanType Culture Collection, Rockville, Maryland, and added to theirpermanent collection of microorganisms at ATCC 39243.

The results of taxonomic studies performed on strain C38,383-RK2indicate that the strain is classified as an atyptical species of thegenus Nocardia. Based on the characteristics indicated below, strainC38,383-RK2 is believed to belong to the species group of Nocardiaaerocolonigenes.

Strain C38,383-RK2 has the following properties:

Morphology

Strain C38,383-RK2 forms unicellular filamentous cells which developinto substrate and aerial mycelia. Both mycelia are long, well branchedand not fragmented into short filaments (0.5 μm in width). Arthrosporesare born in the whole of aerial mycelium. These spores are arranged withintercalation of empty hyphae, or formed as a continuous chain. Like thesporulation of Nocardiopsis dassonvillei, (Intl. J. Syst. Bacteriol. 26:487-493, 1976) the aerial hyphae of strain C38,383 are divided into longsegments which subsequently subdivide into spores of irregular size. Thechains of intercalary or continuous spores are straight or flexuous inshape. Extremely long spore-chains which contain 50 to 100 spores in achain are formed along with short or moderate length of chains. Thespores are cylindrical in shape, 0.5-0.7×0.7-5 μm in size, and have asmooth surface.

Sclerotia are formed on the aerial mycelium, but sporangia, motilespores and whorls are not observed.

Cultural characteristics

Strain C38,383 is an obligately aerobic actinomycete, and grows well inmost agar media. The aerial mycelium is formed abundantly on Czapek'ssucrose-nitrate agar, ISP Medium Nos. 2,4,5 and 7, nutrient agar andBennett's agar, but poorly on glucose-asparagine agar and ISP MediumNos. 3 and 6. The color of aerial mycelium is white, yellowish white orpale yellow. A yellowish pigment is formed in the substrate mycelium,which diffuses slightly into agar medium. This pigment is not apH-indicator. Melanoid pigment is not produced. The culturalcharacteristics are shown in Table 1.

Physiological Characteristics

The optimal growth temperature for strain C38,383 ranges from 28° C. to37° C., and moderate growth is seen at 20° C. and 41° C. No growth isobserved at 7° C. and 45° C. Gelatin and starch are decomposed.Tyrosinase reaction is negative. The growth is inhibited in the presenceof 8% NaCl, but not by lysozyme at 0.01%. Strain C38,383 utilizes mostsugars for growth. The physiological characteristics and utilization ofcarbohydrates are shown in Tables 2 and 3, respectively.

Cell Wall Amino Acid and Whole Cell Sugar Components

The amino acid composition in the cell wall was examined according tothe methods described by Becker et al. (Appl. Microbial. 13: 236-243,1965) and Yamaguchi (J. Bacteriol. 89: 441-453, 1965), and the sugarcomponent in the whole cell hydrolyzate was identified according to theprocedures outlined by Lechevalier and Lechevalier in Biology of theActinomycetes and Related Organisms 11: 78-92, 1976. The cell wall ofstrain C38,383 contains meso-diaminopimelic acid but lacks glycine.Whole cell hydrolyzate shows the presence of glucose, galactose, mannoseand rhamnose. The above-mentioned cell wall composition and whole cellsugar components indicate that the strain C38,383 is an actinomycetespecies of cell wall type IIIC.

Taxonomy

Strain C38,383 was compared with eight genera of order Actinomycetales,including Nocardia, Micropolyspora, Microtetraspora, Nocardiopsis,Saccharopolyspora, Pseudonocardia, Actinomadura and Streptoalloteichus,all of which produce spore-chains on the aerial mycelium and containmesodiiaminopimelic acid in the cell wall. Among these eight genera, thegenus Nocardiopsis is most related to strain C38,383 in the spore-chainand spore morphology, but differs from strain C38,383 in the absence ofgalactose and mannose in the whole cell hydrolyzate.

Gordon et al. (J. Gen. Microbiol. 109: 69-78, 1978) characterized 14taxa of nocardiae based on the physiological properties and the chemicalcomposition in whole cell hydrolyzate. Strain C38,383 was most similarto Nocardia aerocolonigenes in the amino acid and sugar composition inwhole cell hydrolyzate. Therefore, strain C38,383 was compared with thediagnostic physiological properties of N. aerocolonigenes. As shown inTable 4, strain C38,383 was found to be closely related to N.aerocolonigenes but significantly different from Nocardia (Nocardiopsis)dassonvillei. However, all 14 strains of N. aerocolonigenes lack or losethe abilities to form spores and aerial mycelium. Thus, strain C38,383is considered to be a sporogenic species in the taxon of Nocardiaaerocolonigenes.

Strain C38,383 was also found to lose its ability to form aerialmycelium and spores. After five successive transfers, 70% of singleisolates lost these abilities. Such property of strain C38,383 seems tobe similar to the reported variation of Nocardia aerocolonigenes in theformation of spores and aerial mycelium.

                  TABLE 1                                                         ______________________________________                                        Cultural Characteristics of Strain No. C38,383*                               ______________________________________                                        Tryptone-yeast extract                                                                       G**    moderate; floccose, pale                                broth (ISP No. 1)     yellow pellets                                                         D      none                                                    Sucrose-nitrate agar                                                                         G      abundant                                                (Czapek's agar)                                                                              R      strong yellow (84)*** to                                                      vivid yellow (82)                                                      A      moderate, yellowish white                                                     (92) to pale yellow (89)                                               D      dark grayish yellow (91) to                                                   light olive brown (94)                                  Glucose-asparagine agar                                                                      G      poor                                                                   R      white (263)                                                            A      scant, yellowish white (92)                                                   to pale yellow (89)                                                    D      none                                                    Glycerol-asparagine agar                                                                     G      abundant                                                (ISP No. 5)    R      brilliant yellow (83) to                                                      strong yellow (84)                                                     A      abundant, pale yellow (89)                                                    to light yellow (86)                                                   D      yellow gray (93) to grayish                                                   yellow (90)                                             Inorganic salts-starch agar                                                                  G      abundant                                                (ISP No. 4)    R      pale yellow (89) to strong                                                    yellow (84)                                                            A      abundant, white (263) to                                                      yellowish white (92)                                                   D      none                                                    Tyrosine agar  G      abundant                                                (ISP No. 7)    R      brilliant yellow (83) to                                                      strong yellow (84)                                                     A      moderate, pale yellow (89)                                                    to light yellow (86)                                                   D      pale yellow (89)                                        Nutrient agar  G      abundant                                                               R      yellowish white (92) to pale                                                  yellow (89)                                                            A      abundant, white (263)                                                  D      none                                                    Yeast extract-malt extract                                                                   G      abundant                                                agar (ISP No. 2)                                                                             R      brilliant orange yellow (67)                                                  to strong orange yellow (68)                                           A      abundant, yellowish white (92)                                                to pale yellow (89)                                                    D      dark orange yellow (72) to                                                    moderate yellowish brown (77)                           Oat meal agar  G      moderate                                                (ISP No. 3)    R      light yellow (86) to brilliant                                                yellow (83)                                                            A      scant, yellowish white (92) to                                                pale yellow (89)                                                       D      none                                                    Bennett's agar G      abundant                                                               R      brilliant yellow (83) to strong                                               yellow (84)                                                            A      abundant, yellowish white (92)                                                to pale yellow                                                         D      vivid yellow (82)                                       Peptone-yeast extract-iron                                                                   G      moderate                                                agar (ISP No. 6)                                                                             R      pale yellow (89) to light                                                     yellow (86)                                                            A      poor, white (263) to yellowish                                                white (92)                                                             D      none                                                    ______________________________________                                         *observed after incubation at 28° C. for 3 weeks                       **Abbreviation: G = growth; R = reverse color; A = aerial mycelium; D =       diffusible pigment                                                             ***Color and number in parenthesis follow the color standard in Kelly, K     L. & D. B. Judd: ISCCNBS colorname charts illustrated with Centroid           Colors. US Dept. of Comm. Cir. 553, Washington, D.C., No., 1975".        

                  TABLE 2                                                         ______________________________________                                        Physiological Characteristics of Strain No. C38,383                                                       Method or                                         Test        Response        Medium used                                       ______________________________________                                        Range of tempera-                                                                         Maximal growth at                                                                             Bennett's agar                                    ture for growth                                                                           28° C. to 37° C. Moder-                                         ate growth at 20° C. and                                               41° C. No growth at                                                    7° C. and 45° C.                                    Gelatin liquefaction                                                                      Liquefied       1% malt extract,                                                              0.4% yeast ex-                                                                tract, 0.4% glu-                                                              cose, 20% gelatin.                                Starch hydrolysis                                                                         Hydrolyzed      Starch agar plate                                 Reactions in                                                                              Not coagulated and                                                                            Difco skimmed                                     skimmed milk                                                                              completely peptonized                                                                         milk                                              Formation of mela-                                                                        negative        Tyrosine agar,                                    noid pigment                peptone-yeast                                                                 extract-iron                                                                  agar, and tryp-                                                               tone-yeast                                                                    extract broth                                     Tyrosinase reaction                                                                       Negative        Arai's method*                                    Nitrate reduction                                                                         Positive        Czapek's su-                                                                  crose-nitrate                                                                 broth                                                         Positive        0.5% yeast                                                                    extract, 1% glu-                                                              cose, 0.5% KNO.sub.3,                                                         0.1% CaCO.sub.3.                                  Acid tolerance                                                                            Growth at pH 5.0.                                                                             Yeast extract-                                                No growth at pH 4.5.                                                                          malt extract agar                                 NaCl tolerance                                                                            Growth at 7% NaCl or                                                                          Basal medium: 1%                                              less. No Growth at 8%                                                                         yeast extract,                                                NaCl.           2% soluble starch,                                                            1.5% agar.                                        Lysozyme tolerance                                                                        Tolerant.       Trypticase soy                                                Growth at 0.01% broth plus 1.5%                                               lysozyme.       agar.                                             ______________________________________                                         *Arai, T. and Y. Mikami: Chromogenicity of Streptomyces. Appl. Microbiol.     23: 402-406, 1972.                                                       

                  TABLE 3                                                         ______________________________________                                        Carbohydrate Utilization of Strain No. C38,383                                ______________________________________                                               Glycerol   +                                                                  D(-)-Arabinose                                                                           +                                                                  L(+)-Arabinose                                                                           +                                                                  D-Xylose   +                                                                  D-Ribose   +                                                                  L-Rhamnose +                                                                  D-Glucose  +                                                                  D-Galactose                                                                              +                                                                  D-Fructose +                                                                  D-Mannose  +                                                                  L(-)-Sorbose                                                                             -                                                                  Sucrose    +                                                                  Lactose    +                                                                  Melibiose  +                                                                  Trehalose  +                                                                  Raffinose  +                                                                  D(+)-Melezitose                                                                          -                                                                  Soluble starch                                                                           +                                                                  Cellulose  +                                                                  Dulcitol   -                                                                  Inositol   +                                                                  D-Mannitol +                                                                  D-Sorbitol -                                                                  Salicin    +                                                           ______________________________________                                         observed after incubation at 37° C. for 3 weeks                        Basal medium: PridhamGottlieb's inorganic medium                              Abbreviation: +: positive utilization, -: negative utilization           

                  TABLE 4                                                         ______________________________________                                        Comparison of diagnostic physiological properties among                       strain C38,383, Nocardia aerocolonigenes and Nocardiopsis                     dassonvillei                                                                                    Nocardia*   Nocardiopsis*                                              Strain aerocolonigenes                                                                           dassonvillei                                               C38,383                                                                              (14)**      (31)**                                          ______________________________________                                        Decomposition of                                                              Adenine      -        -           +                                           Casein       +        +           +                                           Hypoxanthine +        +           +                                           Tyrosine     +        +           +                                           Urea         -        +           -                                           Xanthine     -        -           +                                           Resistance to:                                                                Lysozyme     +        +           -                                           Rifampin     -        -           -                                           Hydrolysis of:                                                                Aesculin     +        +           -                                           Hippurate    -        V           +                                           Starch       +        +           +                                           Acid         from:                                                            Inositol     +        +           -                                           Lactose      +        +           -                                           Melibiose    +        +           -                                           Raffinose    +        V           -                                           Utilization of:                                                               Benzoate     -        -           -                                           Citrate      +        +           +                                           Mucate       -        -           -                                           Succinate    +        +           +                                           Tartrate     -        -           -                                           Nitrite from nitrate                                                                       +        V           +                                           Survival at 50° C., 8 h                                                             -        V           +                                           ______________________________________                                         +: positive, -: negative, V: 15 to 84% of the strains positive                *Data of Gordon et al. (J. Gen. Microbiol. 109: 69-78, 1978)                  **No. of strains examined                                                

It is to be understood that the present invention is not limited to useof the particular preferred strain C38,383-RK2 described above or toorganisms fully answering the above descriptions. It is especiallyintended to include other 4'-deschlororebeccamycin-producing strains ormutants of the said organism which can be produced by conventional meanssuch as x-radiation, ultraviolet radiation, treatment with nitrogenmustards, phage exposure, and the like.

Preparation of 4'-Deschlororebeccamycin

4'-Deschlororebeccamycin may be produced by cultivating a4'-deschlororebeccamycin-producing strain of Nocardia aerocolonigenes,preferably a strain having the characteristics of Nocardiaaerocolonigenes strain C38,383-RK2 (ATCC 39243) or mutant thereof, undersubmerged aerobic conditions in an aqueous nutrient medium. The organismis grown in a nutrient medium containing an assimilable carbon source,for example, sucrose, lactose, glucose, rhamnose, fructose, mannose,melibiose, glycerol or soluble starch. The nutrient medium should alsocontain an assimilable nitrogen source such as fish meal, peptone,soybean flour, peanut meal, cottenseed meal or corn steep liquor.Nutrient inorganic salts can also be incorporated in the medium. Suchsalts may comprise any of the usual salts capable of providing sodium,potassium, ammonium, calcium, phosphate, sulfate, chloride, bromide,nitrate, carbonate or like ions.

Production of 4'-deschlororebeccamycin can be effected at anytemperature conducive to satisfactory growth of the organism, e.g.20°-41° C., and is conveniently carried out at a temperature of about27° C.

The fermentation may be carried out in flasks or in laboratory orindustrial fermentors of various capacities. When tank fermentation isto be used, it is desirable to produce a vegetative inoculum in anutrient broth by inoculating a small volume of the culture medium witha slant or soil culture or a lyophilized culture of the organism. Afterobtaining an active inoculum in this manner, it is transferredaseptically to the fermentation tank medium for large scale productionof 4'-deschlororebeccamycin. The medium in which the vegetative inoculumis produced can be the same as, or different from, that utilized in thetank as long as it is such that a good growth of the producing organismis obtained.

In general, optimum production of 4'-deschlororebeccamycin is achievedafter incubation periods of about seven days.

4'-Deschlororebeccamycin is a minor product of the fermentation and maybe recovered from the culture medium and isolated in a substantiallypure form according to the multistep procedure described in Example 1below. Thus, the desired 4'-deschlororebeccamycin is found primarily inthe mycelium and recovery from the mycelium may be effected byextraction with an organic solvent such as tetrahydrofuran. Afterreduction of the extract volume a crude solid containing the desired4'-deschlororebeccamycin may be obtained. This crude solid may then besubjected to a multistep purification scheme illustrated in thefollowing flow chart:

    ______________________________________                                         ##STR3##                                                                      ##STR4##                                                                     ______________________________________                                    

Physicochemical Properties of 4'-Deschlororebeccamycin

The physicochemical properties of 4'-deschlororebeccamycin are asfollows:

4'-Deschlororebeccamycin is a yellow amorphous solid having a molecularformula of C₂₇ H₂₂ O₇ N₃ Cl and a molecular weight of 535.8397. It iscomposed of the elements carbon, hydrogen, oxygen, nitrogen andchlorine. Elemental analysis data is as follows:

Calc'd for C₂₇ H₂₂ O₇ N₃ Cl.H₂ O: C, 58.54; H, 4.37; N, 7.58; Found: C,53,43; H, 4.29; N, 7.29.

The high resolution mass spectrum of 4'-deschlororebeccamycin wasdetermined with a Kratos MS-50 spectrometer and FAB ionization. Theobserved mass is as follows:

Calc'd for (M+H)⁺ ion: 536.1224; Found for (M+H)⁺ ion: 536.1188.

4'-Deschlororebeccamycin is insoluble in water and soluble indimethylsulfoxide.

The infrared absorption spectrum of 4'-deschlororebeccamycin whenpelleted in KBr exhibits characteristic bands at the followingfrequencies exhibited in reciprocal centimeters:

3400, 3330, 2930, 1745, 1703, 1575, 1490, 1470, 1458, 1435, 1398, 1380,1330, 1273, 1238, 1140, 1105, 1083, 1050, 1015, 947, 910, 800, 798, 755,738, 670, 665, 633.

The ultraviolet absorption spectrum of 4'-deschlororebeccamycin wasdetermined in methanol (0.03462 g/l) under neutral conditions. Observedabsorption maxima and absorptivities are as follows:

400 nm (8.6), 315 nm (96.5), 290 nm (107.7), 257 nm (sh), 235 nm (76.3).

A proton magnetic resonance spectrum of 4'-deschlororebeccamycindissolved in dimethylsulfoxide was determined with a Bruker WM-360spectrometer operating at 360 MHz and using tetramethylsilane was theinternal standard. The observed chemical shifts (δ values), couplingconstants (J values in Hz) and pattern descriptions are as follows:

11.81 (s, 1H, N8--H), 11.24 (s, 1H, N5'--H), 9.26 (d, J=7.9, 1H, C1--Hor C1'--H), 9.10 (d, J=7.9, 1H, C1--H or C1'--H) 7.77 (d, J=7.9, 1H,C4'--H), 7.63 (m, 2H, C3--H and C3'--H), 7.42 (m, 2H, C2--H and C2'--H),6.91 (d, J=9.4, 1H, C1"--H), 6.30 (bs, 1H, C6"--OH), 5.25 (d, J=5.7, 1H,C3"--OH), 4.91 (d, J=5.7, 1H, C2"--OH), 4.01 (bs, 2H, C6"--H), 3.90 (d,1H, C5"--H), 3.67 (t, 1H, C4"--H), 3.62 (s, 3H, C4"--OCH₃), 3.53 (m, 1H,C2"--H overlaps with H₂ O).

A carbon-13 magnetic resonance spectrum of 4'-deschlororebeccamycindissolved in dimethylsulfoxide was determined with a Bruker WM-360spectrometer operating at 22.5 MHz and using tetramethylsilane as theinternal standard. The observed chemical shifts (ppm values) andassignments are as follows:

    ______________________________________                                        Chemical Shift (ppm)  Assignment                                              ______________________________________                                        170.7                 C7                                                      170.6                 C7'                                                     140.7                 C4a                                                     138.1                 C4a'                                                    130.4                 C5a                                                     129.9                 C5a'                                                    129.4                 C3'                                                     127.3                 C3                                                      125.6                 C5c                                                     124.6                 Cl'                                                     123.9                 Cl                                                      122.8                 C5c'                                                    122.3                 C2                                                      121.1                 C6                                                      120.6                 C2'                                                     119.4                 C6'                                                     119.1                 C5b'                                                    117.4                 C5b                                                     116.4                 C4                                                      112.1                 C4'                                                     83.9                  Cl"                                                     77.6                  C3"                                                     77.0                  C4"                                                     76.6                  C5"                                                     72.1                  C2"                                                     60.0                  OCH.sub.3                                               58.7                  C6"                                                     ______________________________________                                    

Biological Activity of 4'-Deschlororebeccamycin

The antibacterial activity of 4'-deschlororebeccamycin was determinedagainst a number of gram-positive and gram-negative organisms by theserial two-fold agar dilution method. The results are shown in Table 5below in comparison with the activity of rebeccamycin.

                  TABLE 5                                                         ______________________________________                                        Antibacterial Activity of 4'-Deschlororebeccamycin                                           Minimum Inhibitory Concentra-                                                 tion (MIC) (mcg/ml)                                                                         4'-Deschloro-                                    Organism         Rebeccamycin                                                                              rebeccamycin                                     ______________________________________                                        S. pneumoniae                                                                             A9585    >125        32                                           S. pyogenes A9604    >125        32                                           S. faecalis A20688   8           16                                           S. aureus   A9537    0.5         2                                            M. luteus   A9547    0.5         1                                            S. aureus (Pen-Res)                                                                       A9606    >250        >250                                         S. coli     A15119   >250        >250                                         S. coli     A20341-1 >250        >250                                         K. pneumoniae                                                                             A9664    >250        >250                                         K. pneumoniae                                                                             A20468   >250        >250                                         E. cloacae  A9659    >250        >250                                         E. cloacae  A9656    >250        >250                                         P. mirabilis                                                                              A9900    >250        >250                                         P. vulgaris A21559   >250        >250                                         M. morganii A15153   >250        >250                                         P. rettgeri A22424   >250        >250                                         S. marcescens                                                                             A20019   >250        >250                                         P. aeruginosa                                                                             A9843A   >250        >250                                         P. aeruginosa                                                                             A21213   >250        >250                                         List. monocytogenes                                                                       A15121   32          32                                           List. monocytogenes                                                                       A20025   32          63                                           ______________________________________                                    

4'-Deschlororebeccamycin was also tested against the transplanted mouseleukemia P-388 and the results are shown below in Table 6. Themethodology used generally followed the protocols of the National CancerInstitute [Cancer Chemotherapy Rep. Part 3, 3, 1-103 (1972)]. Theessential experimental details are given at the bottom of Table 6.

                  TABLE 6                                                         ______________________________________                                        Effect of 4'-Deschlororebeccamycin on P-388 Leukemia                                                           Average                                                                       weight  Sur-                                           Dose, IP  MST    MST   change, gm                                                                            vivors                               Material  mg/kg/inj Days   % T/C day 5   day 10                               ______________________________________                                        Rebeccamycin                                                                            512       17.0   155   -1.4    6/6                                            256       15.0   136   -0.3    6/6                                            128       14.5   132   0.2     6/6                                            64        15.0   136   0.3     6/6                                            32        13.0   118   -0.6    6/6                                            16        15.0   136   -0.8    6/6                                  4'-Deschloro-                                                                           512       15.5   141   -1.0    4/4                                  rebeccamycin                                                                            256       15.0   136   -1.5    4/4                                            128       17.5   159   -0.6    4/4                                            64        15.0   136   -0.8    4/4                                            32        15.5   141   -0.8    4/4                                            16        18.0   164   -0.9    3/4                                  Control   0.5 ML    11.0   100   0.6     10/10                                ______________________________________                                         Tumor inoculum: 10.sup.6 ascites cells, ip                                    Host: CDF.sub.1 F mice                                                        Treatment: Single injection on day 1 given i.p.                               Evaluation: MST = median survival time                                        Effect: % T/C = (MST treated/MST control) × 100                         Criteria: % T/C >125 considered significant tumor inhibition                  Control: Saline (0.5 ml) given once daily i.p. for 5 days                

As indicated by the antimicrobial and mouse tumor data provided above,4'-deschlororebeccamycin is useful as an antibiotic and also as anantitumor agent for inhibition of mammalian malignant tumors such asP-388 leukemia.

The invention includes within its scope pharmaceutical compositionscontaining an effective antimicrobial or tumor-inhibiting amount of4'-deschlororebeccamycin in combination with an inert pharmaceuticallyacceptable carrier or diluent. Such compositions may also contain otheractive antimicrobial or antitumor agents and may be made up in anypharmaceutical form appropriate for the desired route of administration.Examples of such compositions include solid compositions for oraladministration such as tablets, capsules, pills, powders and granules,liquid compositions for oral administration such as solutions,suspensions, syrups or elixirs and preparations for parenteraladministration such as sterile solutions, suspensions or emulsions. Theymay also be manufactured in the form of sterile solid compositions whichcan be dissolved in sterile water, physiological saline or some othersterile injectable medium immediately before use.

For use as an antimicrobial agent, the 4'-deschlororebeccamycin orpharmaceutical composition thereof is administered so that theconcentration of active ingredient is greater than the minimuminhibitory concentration for the particular organism being treated. Foruse as an antitumor agent, optimal dosages and regimens of4'-deschlororebeccamycin for a given mammalian host can be readilyascertained by those skilled in the art. It will, of course, beappreciated that the actual dose of 4'-deschlororebeccamycin used willvary according to the particular composition formulated, the mode ofapplication and the particular situs, host and disease being treated.Many factors that modify the action of the drug will be taken intoaccount including age, weight, sex, diet, time of administration, routeof administration, rate of excretion, condition of the patient, drugcombinations, reaction sensitivities and severity of the disease.

The following example is provided for illustrative purposes only and isnot intended to limit the scope of the invention, Skellysolve B is acommercially available petroleum solvent (Skelly Oil Co.) comprisingisomeric hexanes and having a boiling point of 60°-69° C. Dicalite isdiatomaceous earth manufactured by Grefco, Inc. Unless otherwiseindicated, all temperatures below are in degrees Centigrade.

EXAMPLE 1 Preparation of 4'-Deschlororebeccamycin A. Fermentation

Nocardia aerocolonigenes strain C38,383-RK2 (ATCC 39243) was maintainedand transferred in test tubes on agar slants of yeast-malt extract agar.This medium consists of 4.0 g glucose, 4.0 g yeast extract, 10 g maltextract and 20 g agar made up to one liter with distilled water. Witheach transfer the agar slant was incubated for seven days at 27° C. Toprepare an inoculum for the production phase, the surface growth fromthe slant culture was transferred to a 500 ml Erlenmeyer flaskcontaining 100 ml of sterile medium consisting of 30 g glucose, 10 g soyflour, 10 g cottonseed embryo meal and 3 g CaCO₃ made up to one literwith distilled water. This vegetative culture was incubated at 27° C.for 48 hours on a Gyrotory tier shaker (Model G53, New BrunswickScientific Co., Inc.) set at 210 rev/min describing a circle with a 5.1cm diameter. Four ml of vegetative culture was transferred to a 500 mlErlenmeyer flask containing 100 ml of sterile production mediumconsisting of 60 g corn starch, 10 g glucose, 15 g linseed meal, 5.0 gautolyzed yeast, 1.0 g FeSO₄.7H₂ O, 1.0 g NH₄ H₂ PO₄, 1.0 g (NH₄)₂ SO₄and 10 g CaCO₃ made up to one liter with distilled water. The productionculture was incubated at 27° C. on a shaker such as used for thevegetative culture. The agitation rate was set at 250 rev/min. Thefermentation was terminated at 168 hours.

B. Isolation

The fermentation broth obtained according to Example 1A is filteredusing a diatomaceous earth filter aid (the filter aid is admixed withthe broth and also used to form a mat). The filtrate is discarded andthe mat extracted with tetrahydrofuran (THF) for 30-60 minutes using0.1-0.2 volumes based on the original broth volume (the THF preferablycontains 0.025% butylated hydroxytoluenes as preservative). The THFextract is filtered and the insolubles discarded. The filtrate isconcentrated in vacuo until almost all the THF is removed. Inert filteraid is then admixed with the concentrate and the resulting mixture isfiltered on a mat of inert filter aid. Air is sucked through the mat forfour hours or more to dry the mat as much as possible.

The mat obtained as described above is then extracted for about 30minutes with enough THF to get a good slurry. The extract is filteredand the mat discarded. The filtrate is concentrated by boiling at oneatmosphere. Hot methanol is simultaneously added as the volume becomeslow. After crystallization of yellow solids begins, the mixture isboiled gently until bumping becomes a problem. The reaction mixture isthen allowed to cool and is chilled to 5°-8° C. The solid product isfiltered, rinsed with cold methanol and dried. This material containingthe desired 4'-deschlororebeccamycin is used in the following separationprocedure.

C. Separation and Purification

Crude solids from Example 1B (336.3 g) were suspended and partiallydissolved in 2.51 of 2 parts chloroform: 1 part methanol and transferredto a 61 round bottom flask. Approximately 1 kg of filter aid (Dicalite)was mixed into the suspension. The mixture was diluted withapproximately 1.51 of Skellysolve B. The resultant slurry wasconcentrated to a powder in vacuo in a rotatory evaporator. This powderwas slurried in 61 of Skellysolve B and packed into a 12 cm o.d.×90 cmflash chromatography column. A bed was formed with pressurized flow (N₂--5.7 psi). The packed column was eluted with pressurized flow with thefollowing elutropic series: 9 liters of Skellysolve B (3 liters fresh+6liters packing solvent); 13 liters of toluene; 12 liters of methylenechloride; 12 liters of ethyl acetate; 18 liters of tetrahydrofuran; and7 liters of methanol. The toluene eluant was evaporated to dryness invacuo in a rotatory evaporator to yield 5.15 g of solid designatedresidue A.

A Glenco Series 3500 Universal LC column (2.67 cm i.d.×75 cm) was packedwith 80 g Woelm silica gel (0.063-0.200 mm) in chloroform. Residue A wasdissolved in 40 ml of chloroform and pumped directly onto the column.Elution commenced with an initial isocratic rinse of 500 ml chloroform.Elution continued with a 41 linear gradient of chloroform to 5 partsmethanol in 95 parts chloroform collecting twenty 200 ml fractions.Fractions 13 to 16 were judged nearly homogeneous. These were pooled andevaporated to dryness to yield 663 mg of residue B.

The Glenco column (2.67 cm i.d.×75 cm) was packed with Baker BondedPhase Octadecyl silica gel (C-18) in methanol. The column wasequilibrated with approximately 2.5 bed volumes of eluant: 3 partsacetonitrile, 3 parts methanol and 4 parts 0.1M ammonium acetate.Residue B in 3 ml of dimethysulfoxide was drawn into the sample loop andpumped onto the column with eluant. Elution commenced while monitoringthe eluant at 280 nm. After an initial 2 liter forerun, forty 50 mlfractions were collected. Based on the UV chromatogram, fractions 9 to32 were pooled. The composite was extracted with 2 liters of chloroform.The lower phase was separated and concentrated to dryness in vacuo in arotatory evaporator. The residue was partially dissolved in 50 ml ofchloroform with sonication. The suspension was added to 1 liter ofSkellysolve B with rapid stirring. The resultant precipitate wascollected by filtration to yield 606 mg of 4'-deschlororebeccamycin.

Further details of the above isolation procedure are set forth below:

Analytical HPLC

The following components were used to construct an analytical HPLCsystem: Waters Associates Model 6000A Solvent Delivery System pump;Varian Varichrom Model VUV-10 uv/vis Detector set at 254 nm 0.1 O.D.;Fisher Recordal Series 5000 Recorder; Waters Associates Model U6Kinjector; Altex Spherisorb ODS (10μ) column (4.6 mm i.d.×25 cm). Thecomponents were connected with 316 stainless steel tubing (1.6 mmo.d.-0.23 mm i.d.). The eluant of 4 parts acetonitrile, 3 partsmethanol, and 3 parts 0.1M ammonium acetate was pumped at 2 ml/min forall analysis. Occasionally, a Hewlett Packard 1040A HPLC Detector Systemwas substituted for the Varian Varichrom VUV-10 Detector.

Thin Layer Chromatography (TLC)

TLC was carried out on Analtech precoated Silica Gel GHLF plates (2.5cm×10 cm 0.25 mm thick layers). The plates were developed in glasscylinders (6.4 cm diameter by 15 cm high) purchased from Whatman, Inc.The tanks were charged with 10 ml of 5 parts methanol-95 partschloroform and allowed to equilibrate prior to introducing the plate.The developed, air dried plates were visualized with 254 nm and 366 nmultraviolet light using either a Chromato-VUE model CC-20 light box(Ultra-Violet Products Inc.) or a model UVSL-58 hand held mineral lightlamp (Ultra-Violet Products Inc.).

Preparative HPLC

The following components were used to construct a medium pressure liquidchromatography system: Fluid Metering, Inc. Model RP-SY 2CSC FMI LabPump; Fluid Metering Inc. Model PD-60-LF FMI Pulse Dampener; a 15 mlsample loop constructed of polypropylene tubing (3.0 mm o.d.×1.5 mmi.d.) wrapped around a cardboard tube (8.65 cm o.d.); Glenco Series 3500Universal LC column (2.67 cm i.d.×75 cm); Instrumentation SpecialtiesCo. Model UA-5 Absorbance/Fluorescence Monitor with a Type 6 opticalunit; Instrumentation Specialties Co. Model 590 Flow Interrupter Value;and an Instrumentation Specialties Co. Model 328 Fraction Collector. Thecomponents were connected with polypropylene and Teflon tubing (3.0 mmo.d.×1.5 mm i.d.) and Glenco multifit connectors and valves in the orderlisted.

The Glenco series 3500 Universal LC column was slurry packed with thedefined absorbent in the designated solvent using standard techniques.The void between the settled bed and tube top was filled with standardOttawa sand. Eluant was pumped at a maximum rate which would not exceed60 psi back pressure (approximately 20 ml/min).

Gradient Elution

A Glenco gradient elution apparatus consisting of two chambers of equaldiameter, height and volume connected in tandem with a Teflon valve wasused for gradient elutions. One chamber served as a mixing chamber andone as a static reservoir. The less polar solvent, chloroform, wasinitially held in the mixing chamber. The more polar solvent 5 partsmethanol in 95 parts chloroform, was held in the static chamber. Tefloncoated magnetic stirring bars (1.0×3.7 cm) were placed in both chambersand driven by Thomas Model 15 Magne-matic stirrers. Eluant was pumpedfrom the mixing chamber to the medium pressure hplc system throughpolypropylene tubing (1.5 mm i.d.×3.0 mm o.d.). As eluant was removedfrom the mixing chamber, the solvent in the static reservoir was allowedto freely replace it, thus creating a linear gradient of eluant.

We claim:
 1. The compound having the formula ##STR5##
 2. A pharmaceutical composition comprising an effective antibacterial amount of 4'-deschlororebeccamycin having the formula ##STR6## in combination with an inert pharmaceutically acceptable carrier or diluent.
 3. A method for therapeutically treating an animal host affected by a bacterial infection, which comprises administering to said host an effective antibacterial dose of 4'-deschlororebeccamycin having the formula ##STR7## 